A number of diseases are characterized by an exaggerated or untoward immune reactivity against harmless antigens. Such diseases include allergies, autoimmune diseases and inflammatory bowel diseases. Normally, immune responses to harmless antigens are suppressed, a mechanism called tolerance. Tolerance to specific antigens, either exogenous or endogenous, may be induced either by mucosal or systemic exposure.
Tolerance occurs because helper T-cells are deleted, paralyzed or suppressed by other T-cells, so called regulatory T-cells.
Allergies
Allergies are defined as enhanced immune reactivity to one or several harmless environmental antigens, so called allergens. In IgE-mediated allergies, the allergic individual mounts an IgE-antibody response to proteins in foodstuffs, pollens, animal dander, etc. The IgE antibodies are produced by plasma cells developed from B-cells with specificity for a certain allergen. To become an IgE-producing plasma cell, the B-cell must receive help from a T-cell which is specific towards the same allergen. Activation of the T-cell by an allergen leads to the production of cytokines which promotes maturation of the B-cell into a plasma cell that produces IgE. The cytokines IL-4 and IL-13 are especially important in this respect. The subset of T-cells that produce such cytokines and help B-cells to become IgE-producing plasma cells, are called “Th2 cells” (Th=T helper cell). They also commonly produce IL-5, a cytokine which promotes maturation of eosinophils in the bone marrow and activation of such eosinophils that arrive to the tissue where an allergic reaction takes place. Once IgE antibodies are formed, they attach to mast cells in the tissues, for example around blood vessels and in the respiratory and gastro-intestinal tracts. When the allergic individual is exposed to the allergen, e.g. via inhalation or ingestion, minute amounts of intact protein allergen is taken up into the circulation, reaches the mast cells and binds to the IgE antibodies. Hereby the mast cell becomes activated and secretes a range of mediators that trigger the allergic reaction leading to symptoms forming disease entities such as hay fever, asthma, urticaria, atopic eczema, food allergy and allergic anaphylaxis.
In young children, the dominant symptom is atopic eczema, manifested as an itchy rash, or food allergy with gastrointestinal symptoms. Later on, the same child may develop hay fever, i.e. an allergic reaction in the nasal mucosa, caused by IgE-mediated hypersensitivity to environmental antigens. Non-allergic individuals do not mount IgE antibody responses to common environmental antigens, or develop a transient and weak IgE response to food antigens which gradually disappears. The propensity to develop allergy is established in the first few years of life (even if the allergy may manifest itself much later), which has led to a number of measures in order to try to prevent allergy development in children. For example, exclusive breast-feeding and avoidance of exposure to allergens has been widely promoted for many years. However, these measures have been completely ineffective, in that only minute amounts of antigen is needed to trigger IgE production. Many infants may, in fact, develop allergies to egg and cow's milk proteins while being exclusively breast-fed. Furthermore, children from families who have avoided pets are no less allergic to cats and dogs than children who have grown up with such pets in the family.
Allergy is much more common in industrialized countries compared to developing countries, which also applies to autoimmune and inflammatory disorders. This has led to the speculation that exposure to microbes in early childhood affords proper maturation of the developing immune system. However, it is not known which types of microbes are important for this to occur. There is an endless variety of bacteria, viruses and parasites, some of which might be important in providing the right type of stimuli to the immune system, others which may be ineffective, or even increase the risk of developing hypersensitivity or inflammation. For example, the microflora of the gastro-intestinal tract consists of several hundred species, some which are aerobic, while most are obligate anaerobes. The colonizing bacteria can be both Gram-positive and Gram-negative which each differ greatly in cell wall structure and their effects on the immune system.
Yoghurts and other traditional fermented food products have been tried both as therapeutic and preventive agents against allergy. Lactobacillus rhamnosus GG was given to children with severe cow's milk allergy and was shown to ameliorate intestinal inflammation and eczema in these patients. Based on these positive effects, Lactobacillus rhamnosus GG was given to mothers during pregnancy and lactation, and to bottle-fed infants in their formula, as a means to prevent development of allergy in their children.
Indeed, children who were exposed to these lactobacilli had less eczema by two and four years of age compared to children who were not exposed to these bacteria. However, it is important to note that there was no reduction in IgE levels or allergy with respiratory symptoms in children who had been exposed to these lactobacilli during infancy (Kalliornàki et al. Lancet. 2001 Apr. 7; 357(9262):1076-9 and Kalliornàki et al. Lancet. 2003 May 31; 361(9372):1869-71).
Staphylococcus aureus Enterotoxins
Certain bacteria produce toxins, i.e. protein molecules with highly damaging potential. Most bacteria which produce toxins are pathogenic, i.e. cause disease. But toxin-producing bacteria may also reside in the normal flora of the respiratory and/or gastrointestinal tracts without causing harm. For example, newborn infants are commonly colonized by toxin-producing Staphylococcus aureus (S. aureus) in their intestines during their first year of life without showing any symptoms from this colonization. The toxins these strains produce: S. aureus enterotoxin A, B, C or D, or TSST-1 (toxic shock syndrome toxin-1) have so called superantigen function. Superantigens have a bifunctional binding capacity: they bind both to the major histocompatility complex II (MHC II) molecule of an antigen-presenting cell and to the T-cell receptor. Whereas a normal antigen only binds to T-cells that have specificity towards just that antigen, the “superantigen” binds to all T-cells that share one certain 8-chain in their receptor, i.e. belongs to a certain V8-family. This means that they bind to and activate a large proportion (10-30%) of the T-cells in human beings or animals, resulting in a massive cytokine production that may lead to shock and severe symptoms, even death. This is the mechanism behind toxic shock syndrome caused by superabsorbent tampons. TSST-1 producing S. aureus may colonize the tampon, produce TSST-1 which is absorbed across the vaginal epithelium and cause shock. A method to prevent the development of superantigen-induced shock may be to expose mucosal surfaces to the particular superantigen prior to challenge, which leads to specific tolerance to that superantigen (but not other antigens). This desensitization has been attributed to production of IL-10 (Collins et al., Infection and Immunity, Vol. 79, No. 5, 2002).
Toxin-producing S. aureus have been implicated in the pathogenesis of eczema, because eczematous skin lesions are often colonized by S. aureus. It has, thus, been suggested that toxins elaborated by S. aureus can worsen the reaction by stimulating T-cells, leading to tissue damage.
However, this ability of superantigens to stimulate T-cells has been suggested as a therapeutic treatment of cancers, infectious and allergic diseases by the employment of the superantigen to activate specific immune responses (U.S. patent no 2001046501 to Howard et al.), and in W003002143 to Antonsson et al. engineered superantigens including staphylococcal enterotoxins and TSST-1 are used in treatment of various forms of cancer. In W09112818 to Lamb et al. superantigens are parenterally administered to reduce the immune response including T-cells in order to prevent or treat rejection reactions, autoimmune disease, allergic disease and harmful responses to infectious agents. The mechanism proposed is via deletion of T-cells or via induced energy of T-cells. However a treatment that results in anergy or deletion of T-cells would not be recommended as prevention for allergy in children since decreased T-cell function would lead to a poor defense against infections.
Regulatory T-Cells (Tregs)
It is believed that allergy, autoimmune and inflammatory disorders are prevented by so called regulatory T-cells (Treg). These cells suppress activation of helper T-cells and thereby down-regulate many types of immune responses. One population of regulatory T-cells, named CD25+ Treg (or CD4+CD25+CTLA-41. T-cells) are CD4-positive T-cells that have a high density of CD25 on their surface with a certain molecule, CTLA-4, in their cytoplasm which functions in intracellular expression. CD25+ Tregs have a capacity to down-regulate the expansion and activation of helper T-cells. Helper T-cells are T-cells which enhance immune responses such as T-cell mediated cytotoxicity, delayed type hypersensitivity and antibody production. Another marker that can be used to identify Tregs is messenger RNA for the gene Foxp3. Mice and humans which have a non-functioning Foxp3 gene develop a syndrome with high IgE levels in the blood, severe eczema, inflammation in the large bowel wall and autoimmune disease in several organs. This strongly suggests that a poor function of Tregs may cause several diseases characterized by untoward or unregulated immune and inflammatory responses: allergy, autoimmunity and inflammatory diseases.
Tregs are produced in the thymus and exit to the periphery in the first days of life in mice. In humans, cells of the Treg phenotype are present at birth, but express lower levels of Foxp3 compared with cells from adults. It has been described that the number and function of CD25+ Treg can be increased by in vitro stimulation with polyclonal activators as well as specific antigens and transfer of these antigen-expanded cells into mice results in delayed development of autoimmune disease in susceptible mice. Repeated injection of the superantigen Staphylococcus aureus enterotoxin A (SEA) into v133- and V138 transgenic mice resulted in potentiated suppressive function of CD25+ Treg as well as induction of suppressive function in CD25″ T-cells (T-cells that do not express CD25 on their surface and which cannot suppress helper T-cell functions). Superantigen administrated in such a way also results in an activation followed by a severe reduction in the number of T-cells in the animal (GrundstrOm et al. Jour. of Immunology, 2003, 170, 5008-5017). This observed activation/reduction together with the fact that superantigen in the blood circulation leads to shock in humans are the main reasons why Staphylococcal enterotoxins administered into the blood are unsuitable for treatment of humans. Mice on the other hand are much more resistant to the adverse effects of superantigens.
Regulatory T-cells, so called Treg, have come into focus recently. As discussed above, Tregs have the ability to down-regulate many types of untoward immune responses, including allergy, autoimmunity and inflammatory bowel disease. Many methods have been designed to expand and activate this cell type in vitro with the purpose to transfer these expanded and activated cells back to the individual from whom they were derived. In this way, severe autoimmune reactions and transplantation rejections may, theoretically, be treated, as indicated from animal experiments. However, it is completely unethical and impossible to do such procedures as preventive measures in small children.
The present invention discloses a solution to this problem by activation of the immune system by mucosal exposure to S. aureus toxins having a superantigen function, in order to expand and activate regulatory T-cells in vivo in early infancy. By this pretreatment, these cells will already be active when the child becomes exposed to potential allergens in the food and environment. Production of IgE antibodies will be prevented and allergic diseases will not be produced in this child. The mucosal exposure ensures that no hazardous effects are produced. The health statuses of the infants included in this study have been thoroughly investigated by continuous registration of symptoms. The children who were colonized by superantigen-producing S. aureus did not exhibit more gastro-intestinal or other symptoms than other children and were, in fact, completely healthy.
As described above, S. aureus and its superantigen production has been regarded as detrimental in development of allergy and it has been suggested that S. aureus should be eradicated in atopic children.